Glutamatergic synapses comprise the major excitatory pathways in brain. A key feature determining the strength of a glutamatergic synapse is the number of postsynaptic AMPA receptors available for activation. We have preliminary evidence that nicotinic stimulation of astrocytes causes them to release a component that specifically recruits AMPA receptors to postsynaptic sites on neurons, rendering these previously silent synapses functional. This is completely unexpected and suggests a new and important mechanism for shaping excitatory circuits. Astrocytes are pervasive and abundant when glutamatergic synapses are first forming during development, and have significant numbers of ?7-containing nicotinic acetylcholine receptors (?7-nAChRs). Spontaneous nicotinic cholinergic activity mediated in part through ?7-nAChRs is also widespread early in development. Our preliminary evidence suggests that it is these ?7-nAChRs that are responsible for the astrocyte effect on AMPA receptors. In view of the early appearance and widespread occurrence of astrocytes and ?7-nAChR signaling, such a mechanism could have profound consequences for shaping network formation and functional output. The first Specific Aim will test in cell culture the synaptogenic effects of components released uniquely by nicotinic stimulation of astrocyte ?7-nAChRs. Pre- and postsynaptic components at glutamatergic synapses will be examined, synaptic function assessed, and a screen of candidate components initiated. The second Specific Aim will test the hypothesis in vivo, using viral constructs and a GFAP-Cre system in mutant mice to determine if astrocyte ?7-nAChRs play a critical role in recruiting postsynaptic AMPA receptors on neurons in the hippocampus. Subsequent experiments will test whether astrocyte ?7-nAChRs also contribute to synaptic plasticity, focusing on locations where previous studies have shown that long-term potentiation results from increased numbers of AMPA receptors. In addition to providing new insight into fundamental processes in the brain, these studies will have serious biomedical implications. Finding that nicotinic signaling influences such basic features as the functionality of glutamatergic synapses may put the system at high risk during development if exposed early on to nicotine, such as through secondhand smoke or maternal milk, either prematurely stimulating or desensitizing this regulatory input. Further, the results will have immediate relevance for pharmaceutical companies globally targeting ?7-nAChRs in drug design without realizing that astrocyte ?7-nAChRs may have this unique and critical role. This R21 project will test the central hypotheses and lay the groundwork for future projects understanding the role of astrocytes and nicotinic signaling in regulating nervous system form and function. PUBLIC HEALTH RELEVANCE: Astrocytes are cells which are nearly 5 times more abundant than neurons in brain and express receptors that can be stimulated by nicotine. Our preliminary results suggest that nicotinic stimulation of astrocytes has profound effects on the formation of glutamatergic synapses, the principal form of excitatory connections between neurons. This proposal will determine when and how this occurs, and in so doing, will not only make major advances in our understanding of brain development but will also have biomedical consequences by revealing circuits and processes vulnerable to nicotine but possibly amenable to therapeutic intervention.